The patent applications cited supra disclose, inter alia., the characterization and preparation of various hormone and hormone-like receptors, including steroid, thyroid and retinoid receptors such as those represented by the glucocorticoid, mineralcorticoid, thyroid, estrogen related and retinoid classes, and specifically, the glucocorticoid, estrogen, aldosterone and retinoic acid receptors themselves. These specific receptors have been the subject of considerable research and form the particular bases for the inventions disclosed and claimed in these patent applications. Similarly, the extant, parallel scientific literature has focused on the specific receptors listed above from among the classes of receptors that exist.
It is known, for example, that the glucocorticoid receptor belongs to a large super-family of ligand-dependent transcription factors that have themselves diverse roles in homeostasis, growth and development. Comparison of complementary DNAs encoding these receptors, as well as mutational analyses of their coding sequences have identified certain functional domains within the molecule that are thought responsible respectively for DNA binding, hormone binding and nuclear localization. See Evans, et al., Science 240, 889 (1988) for a review of this subject matter. In the case of the glucocorticoid receptor, the so-called DNA binding domain spans some sixty-six amino acids and is highly conserved among various species and this domain has been found to be required in order to activate transcription. See Hollenberg, et al., Cell 49, 39 (1987), Miesfeld, et al., Science 236, 423 (1987), Danielsen, et al., Mol.Endo 1, 816 (1987), Kumar, et al., Cell 51, 941 (1987), Gronemeyer, EMBO J. 6, 3985 (1987), and Waterman, et al., Mol,Endo 2, 14 (1988). This region has been found to contain nine invariant cysteines residues and although the contribution of each cysteine residue to overall function is unknown, as is the actual structure formed by this domain, it has been proposed that these cysteine residues coordinate two zinc ions to form two DNA binding, so-called finger domains which result in a ternary structure thought responsible for its localization and binding to the requisite DNA site. See Klug, et al.. Tr.Biochem.Sci 12, 464 (1987), Bens, et al., Cell 52, 1 (1988), and Evans, supra.
In a location nearer the carboxy-terminal end distal from the DNA binding region is the so-called ligand binding domain which has the demonstrated ability to block activity of the receptor in the absence of hormone. Thus, presence of the requisite hormone relieves the inhibition of the receptor to activity. Deletion of this region has been found to produce a hormone-independent transcription activator. See Godowski, et al., Nature 325. 365 (1987), Hollenberg, et al., supra, Kumar, et al., supra. Danielsen et al., supra, and Adler et al., Cell 52, 685 (1988).
In contrast to these two domains, the sequences lying towards the amino-terminal region from the DNA binding domain are poorly understood both as to structure, and particularly, function. This region is extremely variable both in size and in composition amongst the various receptors--See Evans, supra--and may contribute to the heterogeneity of receptor function. See Kumar et al., supra, and Tora et al., 333, 185 (1988).
Despite extensive analysis, some of which having been reported in the scientific literature, the region(s) that determines trans-activation of transcription initiation remains poorly characterized. Trans-activation domains can be defined as polypeptide regions that, when combined with the DNA binding functional domain, increase productive transcription initiation by RNA polymerases. See Sigler, Nature 333, 210 (1988), Brent et al., Cell 43. 729 (1985), Hope et al., Cell 46, 885 (1986), Ma et al., Cell 48, 847 (1987), Ma et al., Cell 51, 113 (1987), Lech et al., Cell 52, 179 (1988), and Hope et al., Nature 333, 635 (1988).
Previous research of the human glucocorticoid receptor by linker scanning mutagenesis identified two regions outside of the DNA binding region having a role in transcription activation. These regions were defined as .tau..sub.1 and .tau..sub.2. Giguere et al., Cell 46, 645 (1986). Further research from these laboratories has also resulted in the report of a co-localization of transactivation and DNA binding functions. See Hollenberg et al., supra. Miesfeld, et al., supra. Danielsen et al., supra, and Waterman et al., supra. As a composite, this research has given rise merely to an emerging picture of an increasingly modular molecule with discrete domains, each contributing to the identified properties of ligand-binding, DNA-binding and trans-activation of transcription. However, until now, the region(s) determining the trans-activation activity, was not at all well understood. Thus, the picture based upon existing research lacks an appreciation of the dynamic nature of the steroid receptors and how the various domains determine the cascade of events initiated by ligand-binding and consummated by promoter-specific transactivation.
Further, although previous research has identified functional "domains", there has been little systematic effort to identify amino acids that contribute to the specific activities of the molecule itself. Thus, the previous identification of steroid receptor transactivation regions resulted only from a demonstrated loss of activity via deletion or insertional mutagenesis, but in no case have the properties of the regions themselves been confirmed in assays that reflect a dominant gain of function.
Thus, Godowski et al., Science 241, 812 (1988), report results that show that the glucocorticoid receptor contains at least one "enhancement domain" other than that overlapping the glucocorticoid response element binding region and that the second domain occupies a region near the receptor amino-terminus. Similarly, Webster et al., Cell 54, 199 (1988) report on an inducible transcription activation function of the estrogen and glucocorticoid receptors, and these researchers speculate that the relative positions of the hormone regions (i.e., ligand and DNA-binding domains) are not important for the transcription induction activity of the receptor. Yet, these researchers admit that they have no definition of the exact location and nature of what they call the hormone-inducible activating domain, to say nothing of its characterization and role in trans-activating potential.
As a starting point for the present invention, Giguere et al., supra, demonstrated loss of activity in the glucocorticoid receptor based upon an assay measuring transcription activity, when random site-directed mutagenesis was performed at several locations of the molecule. As a followup, Hollenberg et al. deleted regions within the molecule, again demonstrating overall loss of transcription activity induced by such removal of stretches of amino acids.
It is an object of the present invention to identify and characterize the domain(s) responsible for trans-activation transcription activity, and the characterization of such domain(s) in respect of amino acid composition and sequence, to explore the functional interaction of the domain(s), if any, with both the DNA-binding and ligand-binding domains of a given receptor, and finally, to exploit such knowledge via the manipulation of such identified and characterized transactivation transcription domain(s) so as to increase the overall transcription activity of the given receptor so manipulated.
The present invention thus provides novel hormone or hormone-like receptors that have been modified by advantage of knowledge of the identity and characterization of the trans-activation transcription activity domain(s), by modifications thereof so as to produce novel, heterologous receptors that have increased activity compared with the parent molecule. It is an object of the present invention to provide novel, heterologous, optionally hybrid receptors having increased trans-activation transcription activity and otherwise having DNA-binding and ligand-binding domains that may be borrowed from various different receptors. It is a further object of the present invention to provide novel assays whereby putative receptor agonists and antagonists can be screened and evaluated for potential commercial exploitation. See also Ptashne, Nature 335, 683 (1988).